DNA double-strand breaks (DSBs) are critical lesions as they can generate genome rearrangements. This project has been focused on the roles of DNA dependent protein complex (DNA-PK) and its interactions with other DNA repair factors involved in early DSB responses. With our extensive and persistent effort during the past funding periods we begin to understand the molecular mechanisms whereby DNA-PK complex acts in DSB sensing, signaling and repair. Our results strongly support the hypothesis that DNA-PK is required for the initial phase of DSB sensing throughout the cell cycle upon DNA damage. Furthermore, the kinase activity of DNA-PKcs plays a pivotal role in modulating the cellular choice of DSB repair pathways between nonhomolgous end joining (NHEJ) and homologous recombination (HR). In this renewal application, we propose to test these hypotheses by employing cellular, molecular and transgenic mouse model approaches. Especially, we will utilize newly established in vivo imaging facility to dissect the temporal relationship among DNA-PK and other DSB sensing complex at DSB sites in live cells. Our research not only will provide a fundamental understanding of the mechanism of DSB repair, but also will unveil mechanism-based targets for the development of potential radiosensitization tools for cancer therapy. The specific aims of the renewal application are: 1) To dissect the mechanism by which DNA-PKcs is activated at sites of DNA DSBs and the role of DNA-PK complex (DNA-PKcs and Ku70/80 heterodimer) in recruiting other NHEJ repair factors in vivo; 2) To test the hypothesis that DNA-PKcs phosphorylation is essential for the NHEJ and also regulates the overall DSB repair machinery by modulating cross talk between NHEJ and HR; 3) To test the hypothesis that ATM and DNA-PKcs are coordinated in sensing and repairing the DNA DSBs in mammalian cells; and 4) To determine the functions of DNA-PKcs phosphorylation and kinase activity in transgenic mouse models.